Peroxisomes are ubiquitous organelles in eukaryotic cells and are responsible for the oxidation of fatty acids, ether lipids, and certain steroids. Along with mitochondria, these organelles segregate toxic reactive oxygen species, such as hydrogen peroxide and superoxide anions, from the rest of the cell to prevent damage to proteins and nucleic acids associated with many diseases, including cancer. To support these functions, the peroxisome must recognize and transport over one hundred distinct proteins across the peroxisomal membrane. Recent studies have established that in contrast to several other cellular organelles, which import proteins in an unfolded state, peroxisomes are capable of importing folded and even oligomeric proteins. Although nearly twenty proteins have been implicated in this process, the mechanistic details of import are still unclear. In addition, many fundamental aspects regarding this pathway have yet to be defined, including the interactions of the receptor with the peroxisomal membrane upon docking, the identity of the translocation channel, the regulation of receptor cycling by ubiquitination, and receptor export from the peroxisome. Therefore, we propose to undertake a biochemical analysis of three critical protein complexes identified in this process in order to elucidate the means by which folded cargo proteins and oligomers are targeted to and transported across the peroxisomal membrane. The specific aims of this proposal are: (1) to characterize the peroxisomal import docking complex and its interactions with the cargo protein and receptor; (2) to isolate the putative ubiquitin ligase complex and investigate the role of ubiquitination in receptor recycling; (3) to elucidate the mechanism of membrane recruitment of the AAA ATPases implicated in receptor export from the peroxisome. This research addresses a central question in cellular biology and is of critical medical relevance, as defects in nearly every step of this import pathway have been linked to the peroxisomal biogenesis disorders including Zellweger syndrome and infantile Refsum's disease. The long-term goal of this work is to develop an in vitro system of peroxisomal protein import, which will allow for the precise determination of how the peroxisome maintains the integrity of its membrane during the import of protein oligomers without leaking dangerous reactive oxygen species that can lead to cellular damage and human disease.